Journal: Oncotarget

Article Title: Hypoxia-inducible factors regulate pluripotency factor expression by ZNF217- and ALKBH5-mediated modulation of RNA methylation in breast cancer cells

doi: 10.18632/oncotarget.11743

Figure Lengend Snippet: A-C. Breast cancer cell lines were exposed to 20% or 1% O 2 for 24 h and NANOG (A), KLF4 (B), and SOX2 (C) mRNA levels were determined by RT-qPCR, relative to 18S rRNA, and normalized to the mean value for MDA-MB-231 cells (MDA231) at 20% O 2 (mean ± SEM; n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001 vs . same cell line at 20% O 2 by Student's t test. D and E. HCC-1954 (D) and MCF-7 (E) subclones, which were stably transfected with an expression vector encoding a non-targeting control (NTC) shRNA, or vector encoding shRNA targeting HIF-1α (sh1α) or HIF-2α (sh2α), or vectors encoding shRNAs targeting both HIF-1α and HIF-2α (DKD), were exposed to 20% or 1% O 2 for 24 h and RT-qPCR was performed to determine NANOG (D) or KLF4 (E) mRNA levels relative to 18S rRNA. The results were normalized to NTC at 20% O 2 (mean ± SEM; n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001 vs . NTC at 20% O 2 ; # P < 0.05, ## P < 0.01, ### P < 0.001 vs . NTC at 1% O 2 by ANOVA. F . ZR75.1 cells treated with vehicle or digoxin (200 nM) were exposed to 20% or 1% O 2 for 24 h and SOX2 mRNA was measured (mean ± SEM; n = 3). * P < 0.05, ** P < 0.01 vs . NTC at 20% O2; ### P < 0.001 vs . NTC at 1% O2 by ANOVA. G and H . NTC and DKD subclones of HCC-1954 (G) and MCF-7 (H) were exposed to 20% or 1% O 2 for 48 h, whole cell lysates were prepared, and immunoblot assays were performed to analyze HIF-1α, HIF-2α, NANOG and KLF4 protein expression. Actin was also analyzed as a loading control. I. ZR75.1 cells were treated with vehicle or digoxin (200 nM), exposed to 20% or 1% O 2 for 48 h, and HIF-1α, NANOG and SOX2 immunoblot assays were performed.

Article Snippet: Blots were probed with HIF-1α (#610959, BD Biosciences, San Jose, CA), HIF-2α (#NB100-122, Novus Biologicals, Littleton, CO), ALKBH5 (#NBP1-82188, Novus Biologicals), NANOG (#NB100-588, Novus Biologicals), KLF4 (#NBP1-83940, Novus Biologicals), SOX2 (NBP1-42823, Novus Biologicals), ZNF217 (#NBP1-78189, Novus Biologicals), and Actin (#sc-1616, Santa Cruz Biotechnology, Dallas, TX) antibodies.

Techniques: Quantitative RT-PCR, Stable Transfection, Transfection, Expressing, Plasmid Preparation, Control, shRNA, Western Blot

Journal: Oncotarget

Article Title: Hypoxia-inducible factors regulate pluripotency factor expression by ZNF217- and ALKBH5-mediated modulation of RNA methylation in breast cancer cells

doi: 10.18632/oncotarget.11743

Figure Lengend Snippet: Induction of BCSCs, pluripotency factors and m 6 A-regulating proteins by hypoxia

Article Snippet: Blots were probed with HIF-1α (#610959, BD Biosciences, San Jose, CA), HIF-2α (#NB100-122, Novus Biologicals, Littleton, CO), ALKBH5 (#NBP1-82188, Novus Biologicals), NANOG (#NB100-588, Novus Biologicals), KLF4 (#NBP1-83940, Novus Biologicals), SOX2 (NBP1-42823, Novus Biologicals), ZNF217 (#NBP1-78189, Novus Biologicals), and Actin (#sc-1616, Santa Cruz Biotechnology, Dallas, TX) antibodies.

Techniques:

Journal: Frontiers in Immunology

Article Title: GM-CSF Inhibits c-Kit and SCF Expression by Bone Marrow-Derived Dendritic Cells

doi: 10.3389/fimmu.2017.00147

Figure Lengend Snippet: Granulocyte-macrophage colony-stimulating factor (GM-CSF) modulates c-kit and stem cell factor (SCF) expression by BM-derived DCs (BMdDCs) . (A–C) Effect of GM-CSF and cell density on c-kit expression by BMdDCs. BMdDCs were plated in 24-well plates and cultured for 2 days in complete Opti-MEM medium in four different conditions, that is at either 2 × 10 5 or 1.2 × 10 5 cell/well, and either with or without (w/o) GM-CSF at 20 ng/ml, as indicated. Cells were stained with fluorochrome-conjugated monoclonal antibodies (mAbs) and analyzed by flow cytometry. (A) Typical flow cytometric profiles, showing CD40 and MHCII expression by BMdDCs. Numbers represent percentages of cells in the indicated regions. (B) Typical histograms showing c-kit expression by MHCII int CD40 int and MHCII hi CD40 hi BMdDCs, gated as in (A) . Solid lines represent c-kit staining profiles, dashed lines represent isotype control mAb. Numbers indicate c-kit median fluorescence intensity (MFI) values. (C) Summary of c-kit expression results obtained from MHCII hi CD40 hi BMdDCs, gated as in (A) . c-kit MFI from individual samples and average values (bar). (D,E) Effect of GM-CSF on SCF expression by BMdDCs. (D) Cell lysates were prepared from day 0 BMdDCs and BMdDCs cultured for 2 days in 24-well plates at 1.2 × 10 5 cell/well in complete Opti-MEM medium with or w/o GM-CSF at 20 ng/ml, as indicated. SCF protein expression was analyzed by ELISA, testing 25 µg of cell lysate in 100 µl/well. Data are expressed as picograms per milliliter. Individual results from three experiments and average values (bar) are shown. (E) Day 0 and day 2 BMdDCs cultured in 24-well plates at 1.2 × 10 5 cell/well with or w/o GM-CSF at 20 ng/ml were analyzed by Real-Time PCR in triplicates. SCF mRNA expression was calculated relative to hprt1 in arbitrary units. For each experiment, day 2 c-kit/hprt1 levels were normalized with day 0. In (A,B) representative data of N = 4 experiments, in (C) N = 4 experiments, in (D) N = 3 experiments, in (E) mean ± SD of four experiments (* P ≤ 0.05; ** P ≤ 0.01).

Article Snippet: BMdDC culture supernatants (100 μl/well) and BMdDC lysates (25 μg of cell lysate/well) were tested by mouse SCF ELISA kit (Boster Immunoleader, Pleasanton, CA, USA).

Techniques: Expressing, Derivative Assay, Cell Culture, Staining, Bioprocessing, Flow Cytometry, Control, Fluorescence, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

Journal: Autophagy

Article Title: Kit-mediated autophagy suppression driven by a viral oncoprotein emerges as a crucial survival mechanism in Merkel cell carcinoma

doi: 10.1080/15548627.2025.2477385

Figure Lengend Snippet: MCPyV truncated LT induces paranuclear retention and stabilization of KIT. (A) Immunofluorescence detection of KIT (green) and MCPyV LT (CM2B4, red) or sT (CM8E6, red) in KIT-HEK293 cells transfected with MCPyV T-antigens or vector control ( n = 6). (B) Top : illustration of the expression constructs of LT339 and the VPS39-interaction defective mutant LT339 W209A . Bottom : Representative images showing the effect of LT339 and LT339 W209A mutant on localization of KIT (green). LT was detected by CM2B4 (red). ( n = 3) (C) Immunoblots showing the effect of the LT339 and LT339 W209A on KIT expression. The quantification of KIT level is shown below the immunoblots ( n = 9). (D) Top : immunoblot analysis of the effect LT339 and LT339 W209A on KIT protein stability in the presence of cycloheximide (CHX) up to 4 h. Bottom : quantification of KIT protein stability after normalization to 0 h time point ( n = 4). (E) Immunoblots showing KIT protein stability in KIT-HEK293 cells transfected with LT339, LT339 W209A or plasmid control (CTR) treated with KIT ligand (KITLG, 100 ng/mL) or solvent control (PBS) in the presence of CHX (100 μg/mL). (F) Quantification of KIT protein stability after normalization to 0 h time point ( n = 5). Solid lines represent PBS control (KITLG − ) and dotted lines represent KITLG treatment (KITLG + ). (A and B) Nuclei were stained by DAPI (blue). Scale bar: 10 μm. Numbers below the images refer to the proportion of cells with KIT paranuclear dot-like staining to the total number of cells analyzed. (C, D and F) Error bars represent mean ± SEM. * p < 0.05, *** p < 0.001, ns = not significant were calculated by one-way ANOVA with post-hoc Tukey’s test (C), two-way ANOVA (D) or two-way ANOVA with post-hoc Bonferroni’s test (F).

Article Snippet: For KITLG induced KIT degradation experiments, KIT-293 cells were transfected with LT339, LT339 W209A or CTR for 48 h, followed by addition of CHX (100 μg/mL) and KITLG (100 ng/mL; Proteintech Group, HZ-1024) or PBS.

Techniques: Immunofluorescence, Transfection, Plasmid Preparation, Control, Expressing, Construct, Mutagenesis, Western Blot, Solvent, Staining